Glycine blocks the increase in intracellular free Ca2+ due to vasoactive mediators in hepatic parenchymal cells

Recently, glycine has been shown to prevent liver injury after endotoxin treatment in vivo. We demonstrated that ethanol and endotoxin stimulated Kupffer cells to release PGE(2), which elevated oxygen consumption in parenchymal cells. Because glycine has been reported to protect renal tubular cells, isolated hepatocytes, and perfused livers against hypoxic injury, the purpose of this study was to determine whether glycine prevents increases in intracellular free Ca(2+) concentration ([Ca(2+)](i)) in hepatic parenchymal cells by agonists released during stress, such as with PGE(2) and adrenergic hormones. Liver parenchymal cells isolated from female Sprague-Dawley rats were cultured for 4 h in DMEM/F12 medium, and [Ca(2+)](i) in individual cells was assessed fluorometrically using the fluorescent calcium indicator fura 2. PGE(2) caused a dose-dependent increase in [Ca(2+)](i) from basal values of 130 +/- 10 to maximal levels of 434 +/- 55 nM. EGTA partially prevented this increase, indicating that either extracellular calcium or agonist binding is Ca(2+) dependent. 8-(Diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an agent that prevents the release of Ca(2+) from intracellular stores, also partially blocked the increase in [Ca(2+)](i) caused by PGE(2), suggesting that intracellular Ca(2+) pools are involved. Together, these results are consistent with the hypothesis that both the intracellular and extracellular Ca(2+) pools are involved in the increase in [Ca(2+)](i) caused by PGE(2). Interestingly, glycine, which activates anion (i.e., chloride) channels, blocked the increase in [Ca(2+)](i) due to PGE(2) in a dose-dependent manner. Low-dose strychnine, an antagonist of glycine-gated chloride channel in the central nervous system, partially reversed the inhibition by glycine. When extracellular Cl(-) was omitted, glycine was much less effective in preventing the increase in [Ca(2+)](i) due to PGE(2). Phenylephrine, an alpha(1)-type adrenergic receptor agonist, also increased [Ca(2+)](i), as expected, from 159 +/- 20 to 432 +/- 43 nM. Glycine also blocked the increase in [Ca(2+)](i) due to phenylephrine, and the effect was also reversed by low-dose strychnine. Together, these data indicate that glycine rapidly blocks the increase in [Ca(2+)](i) in hepatic parenchymal cells due to agonists released during stress, most likely by actions on a glycine-sensitive anion channel and that this may be a major aspect of glycine-induced hepatoprotection.     

Presynaptic Glycine Receptors Influence Plasma Membrane Potential and Glutamate Release

Glycine is a classical inhibitory neurotransmitter however presynaptic glycine receptors have rather depolarizing action. Reasons for latter phenomenon are unknown. In the present paper we have investigated how glycine influences cytosolic chloride level monitored by fluorescent dye SPQ, membrane potential monitored by fluorescent dye DiSC3(5) and [¹⁴C]-glutamate release in synaptosomes. We estimated that cytosolic chloride concentration in synaptosomes was about 52 ± 1 mM. Glycine (1 mM) induced chloride efflux and caused slow plasma membrane depolarization. Chloride efflux was almost completely blocked by 100 μM strychnine whilst glycine-induced depolarization was only partially. We also showed that 1 mM glycine induced [¹⁴C]-glutamate release via a strychnine-insensitive pathway. Hence we have concluded that glycine was able to induce two independent effects in synaptosomes: (1) Chloride efflux with following depolarization. This efflux was sensitive to strychnine and thereby is probably conducted through glycine-gated ion channels. (2) Glutamate release seems to be mediated by glycine transporters.     

Mitochondria buffer NCX-mediated Ca2+-entry and limit its diffusion into vascular smooth muscle cells

The reverse-mode of the Na(+)/Ca(2+)-exchanger (NCX) mediates Ca(2+)-entry in agonist-stimulated vascular smooth muscle (VSM) and plays a central role in salt-sensitive hypertension. We investigated buffering of Ca(2+)-entry by peripheral mitochondria upon NCX reversal in rat aortic smooth muscle cells (RASMC). [Ca(2+)] was measured in mitochondria ([Ca(2+)](MT)) and the sub-plasmalemmal space ([Ca(2+)](subPM)) with targeted aequorins and in the bulk cytosol ([Ca(2+)](i)) with fura-2. Substitution of extracellular Na(+) by N-methyl-d-glucamine transiently increased [Ca(2+)](MT) ( approximately 2microM) and [Ca(2+)](subPM) ( approximately 1.3microM), which then decreased to sustained plateaus. In contrast, Na(+)-substitution caused a delayed and tonic increase in [Ca(2+)](i) (<100nM). Inhibition of Ca(2+)-uptake by the sarcoplasmic reticulum (SR) (30microM cyclopiazonic acid) or mitochondria (2microM FCCP or 2microM ruthenium red) enhanced the elevation of [Ca(2+)](subPM). These treatments also abolished the delay in the [Ca(2+)](i) response to 0Na(+) and increased its amplitude. Extracellular ATP (1mM) caused a peak and plateau in [Ca(2+)](i), and only the plateau was inhibited by KB-R7943 (10microM), a selective blocker of reverse-mode NCX. Evidence for ATP-mediated NCX-reversal was also found in changes in [Na(+)](i). Mitochondria normally exhibited a transient elevation of [Ca(2+)] in response to ATP, but inhibiting the mitochondrial NCX with CGP-37157 (10microM) unmasked an agonist-induced increase in mitochondrial Ca(2+)-flux. This flux was blocked by KB-R7943. In summary, mitochondria and the sarcoplasmic reticulum co-operate to buffer changes in [Ca(2+)](i) due to agonist-induced NCX reversal.     

Cholecystokinin-8 induces intracellular calcium signaling in cultured myenteric neurons from neonatal guinea pigs

The responsiveness of cultured myenteric neurons to cholecystokinin (CCK-8) was examined using fura-2-based digital microfluorimetric measurement of intracellular calcium ([Ca(2+)](i)). CCK-8 (10(-10)-10(-6)M) evoked concentration-dependent increases in percentage of neurons responding (8-52%) and delta[Ca(2+)](i) (76-169 nM). Gastrin (1 microM) also induced an increase in [Ca(2+)](i) in 29+/-6% of neurons (delta[Ca(2+)](i): 71+/-3 nM). L-364,718, an antagonist for the CCK-A receptor, blocked [Ca(2+)](i) response to CCK-8. Removal of extracellular calcium eliminated CCK-induced [Ca(2+)](i) increments, as did the addition of the calcium channel inhibitors nickel (1mM) and lanthanum (5mM). Nifedipine (1-50 microM) dose-dependently attenuated CCK-caused [Ca(2+)](i) responses. CCK evokes [Ca(2+)](i) signaling in myenteric neurons by the influx of extracellular calcium, likely through L-type calcium channels.     

Mechanism of Inhibition of N-Methyl-d-Aspartate-Stimulated Increases in Free Intracellular Ca2+ Concentration by Ethanol

Dissociated brain cells were isolated from newborn rat pups and loaded with fura-2. These cells were sensitive to low N-methyl-D-aspartate (NMDA) concentrations with EC50 values for NMDA-induced intracellular Ca2+ concentration ([Ca2+]i) increases of approximately 7-16 microM measured in the absence of Mg2+. NMDA-stimulated [Ca2+]i increases could be observed in buffer with Mg2+ when the cells were predepolarized with 15 mM KCl prior to NMDA addition. Under these predepolarized conditions, 100 mM ethanol inhibited 25 microM NMDA responses by approximately 50%, which was similar to the ethanol inhibition observed in buffer without added Mg2+. Ethanol did not alter [Ca2+]i prior to NMDA addition. In the absence of Mg2+, 50 and 100 mM ethanol did not significantly alter the EC50 value for NMDA, but did inhibit NMDA-induced increases in [Ca2+]i in a concentration-dependent manner at 4, 16, 64, and 256 microM NMDA. Whereas NMDA-induced increases in [Ca2+]i were dependent on extracellular Ca2+ and were inhibited by Mg2+, the ability of 100 mM ethanol to inhibit 25 microM NMDA responses was independent of the external Ca2+ or Mg2+ concentrations. Glycine (1, 10, and 100 microM) enhanced 25 microM NMDA-induced increases in [Ca2+]i by approximately 50%. Glycine (1-100 microM) prevented the 100 mM ethanol inhibition of NMDA-stimulated [Ca2+]i observed in the absence of exogenous glycine. MK-801 (25-400 nM) inhibited 25 microM NMDA-stimulated rises in [Ca2+]i in a concentration-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Superoxide responses to formyl-methionyl-leucyl-phenylalanine in primed neutrophils. Role of intracellular and extracellular calcium.

For superoxide (O2-) responses of human neutrophils stimulated by FMLP, experiments were designed to assess the requirement of extracellular calcium [( Ca2+]o) for priming of O2- responses by platelet-activating factor (PAF), PMA, or ionomycin. Although priming by PMA did not require [Ca2+]o, there was, as expected, a requirement for [Ca2+]o for the optimal priming effects of PAF and ionomycin. The ED50 value for [Ca2+]o in the priming function of PAF was 105 microM. The [Ca2+]o-dependent priming with ionomycin was bimodal with two ED50 values for [Ca2+]o of 90 microM and 3.2 mM. Optimal priming by PAF required at least 4-min exposure of cells to [Ca2+]o. Cells primed by PAF exhibited faster initial rates of O2-production after addition of FMLP, but the duration of O2- production was not prolonged. Whereas PAF-primed responses to FMLP are usually associated with increases in intracellular calcium [( Ca2+]i) after addition of FMLP, two conditions were found in which O2- responses to FMLP in PAF-primed cells occurred in the absence of any detectable increase in [Ca2+]i. When cells were loaded with the calcium chelator, bis-(O-aminophenoxy)-ethane-H,N,N',N'-tetraacetic acid, and then primed with PAF, normal amounts of inositol 1,4,5-trisphosphate were formed, but no increase in [Ca2+]i occurred after addition of FMLP even though the cells exhibited a fully primed O2- response; in Ca2(+)-depleted and ionomycin-permeabilized cells that were primed with PAF and then stimulated with FMLP, O2- was generated in amounts comparable to reference control (primed) cells, but there was suppressed production of inositol 1,4,5-trisphosphate and no increase in [Ca2+]i after addition of FMLP to PAF-primed cells. These data confirm the requirement of [Ca2+]o for optimal priming of neutrophils by PAF and ionomycin (but not cells primed by PMA) and indicate that, under certain conditions, generation of O2- in response to FMLP in PAF-primed neutrophils can occur independent of any increase in [Ca2+]i.     

Effect of diethylstilbestrol (DES) on intracellular Ca(2+) levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Glucose-insensitivity induced by Ca(2+) toxicity in islet beta-cells and its prevention by PACAP

The present study examined whether a sustained increase in cytosolic Ca(2+) concentration ([Ca(2+)](i)) causes glucose-insensitivity in beta-cells and whether it could be modulated by pituitary adenylate cyclase-activating polypeptide (PACAP), a pancreatic insulinotropin. Rat single beta-cells were cultured for 2 days with sustained increases in [Ca(2+)](i), followed by determination of the [Ca(2+)](i) response to glucose (8.3 mM) as monitored with fura-2. High K(+) (25 mM) produced sustained increases in [Ca(2+)](i) in beta-cells, which were inhibited by nifedipine, a Ca(2+) channel blocker. After culture with high K(+), the incidence and amplitude of [Ca(2+)](i) responses to glucose were markedly reduced. This glucose-insensitivity was prevented by the presence of nifedipine or PACAP-38 (10(-13) M and 10-9) M) in high K(+) culture. PACAP-38 attenuated high K(+)-induced [Ca(2+)](i) increases. In conclusion, sustained increases in [Ca(2+)](i) induce glucose-insensitivity (Ca(2+) toxicity in beta-cells) and it is prevented by PACAP possibly in part due to its Ca(2+)-reducing capacity.     

Human umbilical vein endothelial cells (HUVECs) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia

Bleb formation is an early event of cellular damage observed in a variety of cell types upon hypoxia. Although we previously found that the [Ca(2+)](i) rise before bleb formation only at the same loci of HUVECs upon hypoxia (localized [Ca(2+)](i) rise), the mode of the [Ca(2+)](i) rise remains ill-defined. In order to clarify the mechanisms causing the localized [Ca(2+)](i) rise in hypoxia challenged HUVECs, we studied the effects of several Ca(2+) channel blockers or a Ca(2+) chelator, EGTA, which reduces extracellular Ca(2+) concentration on the hypoxia-induced localized [Ca(2+)](i) rise and bleb formation by employing a confocal laser scanning microscopy (CLSM). After the initiation of hypoxia, [Ca(2+)](i) rose gradually in a localized fashion up to 15 min, which was associated with bleb formation at the same loci. The maximal [Ca(2+)](i) rise was 435 +/- 84 nM at the loci of bleb formation. Ca(2+) channel blockers including Ni(2+) (non-specific, 1 mM), nifedipine (L type, 10 microM), nicardipine (L + T type, 10 microM), and cilnidipine (L + N type, 10 microM) did not inhibit either the localized [Ca(2+)](i) rise or bleb formation. Although both the localized [Ca(2+)](i) rise and bleb formation were inhibited by lowering extracellular Ca(2+) concentration below 100 nM, a diffuse [Ca(2+)](i) rise through the cytoplasm remained without bleb formation, which was inhibited by a phospholipase C (PLC) inhibitor, U73122. In conclusion, hypoxia causes both the Ca(2+) mobilization and the Ca(2+) influx in HUVECs and the Ca(2+) influx through unknown Ca(2+) channels is responsible for the localized [Ca(2+)](i) rise integral to bleb formation.     

Paraxanthine, a caffeine metabolite, dose dependently increases [Ca(2+)](i) in skeletal muscle.

It was hypothesized that the caffeine derivative paraxanthine results in subcontracture increases in intracellular calcium concentration ([Ca(2+)](i)) in resting skeletal muscle. Single fibers obtained from mouse flexor digitorum brevis were loaded with a fluorescent Ca(2+) indicator, indo 1-acetoxymethyl ester. After a stable baseline was recorded, the fiber was superfused with physiological salt solution (Tyrode) containing 0.5, 1.0, 2.5, or 5 mM paraxanthine, resulting in [Ca(2+)](i) increases of 6.4 +/- 2.5, 9.7 +/- 3.6, 26.8 +/- 11.7, and 39.6 +/- 9.6 nM, respectively. The increases in [Ca(2+)](i) were transient and were also observed with exposure to 5 mM theophylline and theobromine. Six fibers were exposed to 5 mM paraxanthine followed by 5 mM paraxanthine in the presence of 10 mM procaine (sarcoplasmic reticulum Ca(2+) release channel blocker). There was no increase from baseline [Ca(2+)](i) when fibers were superfused with paraxanthine and procaine, suggesting that the sarcoplasmic reticulum is the primary Ca(2+) source in the paraxanthine-induced response. In separate experiments, intact flexor digitorum brevis (n = 13) loaded with indo 1-acetoxymethyl ester had a significant increase in [Ca(2+)](i) with exposure to 0.01 mM paraxanthine. It is concluded that physiological and low pharmacological concentrations of paraxanthine result in transient, subcontracture increases in [Ca(2+)](i) in resting skeletal muscle, the magnitude of which is related to paraxanthine concentration.     

Modulation of early [Ca2+]i rise in metabolically inhibited endothelial cells by xestospongin C

When energy metabolism is disrupted, endothelial cells lose Ca(2+) from endoplasmic reticulum (ER) and the cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases. The importance of glycolytic energy production and the mechanism of Ca(2+) loss from the ER were analyzed. Endothelial cells from porcine aorta in culture and in situ were used as models. 2-Deoxy-D-glucose (2-DG, 10 mM), an inhibitor of glycolysis, caused an increase in [Ca(2+)](i) (measured with fura 2) within 1 min when total cellular ATP contents were not yet affected. Stimulation of oxidative energy production with pyruvate (5 mM) did not attenuate this 2-DG-induced rise of [Ca(2+)](i), while this maneuver preserved cellular ATP contents. The inhibitor of ER-Ca(2+)-ATPase, thapsigargin (10 nM), augmented the 2-DG-induced rise of [Ca(2+)](i). Xestospongin C (3 microM), an inhibitor of D-myo-inositol 3-phosphate [Ins(3)P]-sensitive ER-Ca(2+) release, abolished the rise. The results demonstrate that the ER of endothelial cells is very sensitive to glycolytic metabolic inhibition. When this occurs, the ER Ca(2+) store is discharged by opening of the Ins(3)P-sensitive release channel. Xestospongin C can effectively suppress the early [Ca(2+)](i) rise in metabolically inhibited endothelial cells.     

Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca(2+) and activation of external Ca(2+) entry in intact cells

The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space.     

Gonadotropin-releasing hormone-stimulated sperm binding to the human zona is mediated by a calcium influx

The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca(2+)-free medium and in the presence of Ca(2+) channel antagonists. We also examined the GnRH effect on the intracellular free Ca(2+) concentration ([Ca(2+)](i)). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca(2+) was present in the medium. In Ca(2+)-free medium or in the presence of 400 nM nifedipine, 80 microM diltiazem, or 50 microM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca(2+)](i) in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca(2+)](i) was fast and transient, from a basal [Ca(2+)](i) of 413 +/- 22 nM to a peak value of 797 +/- 24 nM. The GnRH-induced increase in [Ca(2+)](i) was entirely due to a Ca(2+) influx from the extracellular medium because the increase in [Ca(2+)](i) was blocked by the Ca(2+) chelator EGTA and by the Ca(2+) channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca(2+) influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca(2+) influx but inhibited the progesterone-activated Ca(2+) influx. Finally, the GnRH-induced Ca(2+) influx was blocked by two specific GnRH antagonists, Ac-D-Nal(1)-Cl-D-Phe(2)-3-Pyr-D-Ala(3)-Arg(5)-D-Glu(AA)(6)-GnRH and Ac-(3,4)-dehydro-Pro(1),-p-fluoro-D-Phe(2), D-Trp(3,6)-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca(2+)](i) through T-type, voltage-operated calcium channels.     

Glycine reduces platelet aggregation

It has been demonstrated that a wide variety of white blood cells and macrophages (i.e. Kupffer cells, alveolar and peritoneal macrophages and neutrophils) contain glycine-gated chloride channels. Binding of glycine on the receptor stimulates Cl^? influx causing membrane hyperpolarization that prevents agonist-induced influx of calcium. Since platelet-aggregation is calcium-dependent, this study was designed to test the hypothesis that glycine would inhibit platelet aggregation. Rats were fed diets rich of glycine for 5 days, while controls received isonitrogenous valine. The bleeding time and ADP- and collagen-induced platelet aggregation were measured. Dietary glycine significantly increased bleeding time about twofold compared to valine-treated controls. Furthermore, the amplitude of platelet aggregation stimulated with ADP or collagen was significantly decreased in whole blood drawn from rats fed 2.5 or 5 % dietary glycine by over 50 %. Addition of glycine in vitro (1–10 mM) also blunted rat platelet aggregation in a dose-dependent manner. Strychnine, a glycine receptor antagonist, abrogated the inhibitory effect of glycine on platelet-aggregation in vitro suggesting the glycine works via a glycine receptor. Glycine also blunted aggregation of human platelets. Further, the glycine receptor was detected in both rat and human platelets by western blotting. Based on these data, it is concluded that glycine prevents aggregation of platelets in a dose-dependent manner via mechanisms involving a glycine receptor.     

Effect of clomiphene on Ca(2+) movement in human prostate cancer cells

The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of PC3 human prostate cancer cells was explored by using fura-2 as a Ca(2+) indicator. Clomiphene at concentrations between 10-50 microM increased [Ca(2+)](i) in a concentration-dependent manner. The [Ca(2+)](i) signal was biphasic with an initial rise and a slow decay. Ca(2+) removal inhibited the Ca(2+) signal by 41%. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with clomiphene in Ca(2+)-free medium, confirming that clomiphene induced Ca(2+) entry. In Ca(2+)-free medium, pretreatment with 50 microM brefeldin A (to permeabilize the Golgi complex), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca(2+) pump), and 2 microM carbonylcyanide m-chlorophenylhydrazone (to uncouple mitochondria) inhibited 25% of 50 microM clomiphene-induced store Ca(2+) release. Conversely, pretreatment with 50 microM clomiphene in Ca(2+)-free medium abolished the [Ca(2+)](i) increase induced by brefeldin A, thapsigargin or carbonylcyanide m-chlorophenylhydrazone. The 50 microM clomiphene-induced Ca(2+)release was unaltered by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). Trypan blue exclusion assay suggested that incubation with clomiphene (50 microM) for 2-15 min induced time-dependent decrease in cell viability by 10-50%. Collectively, the results suggest that clomiphene induced [Ca(2+)](i) increases in PC3 cells by releasing store Ca(2+) from multiple stores in an phospholipase C-independent manner, and by activating Ca(2+) influx; and clomiphene was of mild cytotoxicity.     

Membrane potential dependent modulations of calcium oscillations in insulin-secreting INS-1 cells

This study was undertaken to examine the role of K(+) channels on cytosolic Ca(2+) ([Ca(2+)](i)) in insulin secreting cells. [Ca(2+)](i) was measured in single glucose-responsive INS-1 cells using the fluorescent Ca(2+) indicator Fura-2. Glucose, tolbutamide and forskolin elevated [Ca(2+)](i) and induced [Ca(2+)] oscillations. Whereas the glucose effect was delayed and observed in 60% and 93% of the cells, in a poorly and a highly glucose-responsive INS-1 cell clone, respectively, tolbutamide and forskolin increased [Ca(2+)](i) in all cells tested. In the latter clone, glucose induced [Ca(2+)](i) oscillations in 77% of the cells. In 16% of the cells a sustained rise of [Ca(2+)](i) was observed. The increase in [Ca(2+)](i) was reversed by verapamil, an L-type Ca(2+) channel inhibitor. Adrenaline decreased [Ca(2+)](i) in oscillating cells in the presence of low glucose and in cells stimulated by glucose alone or in combination with tolbutamide and forskolin. Adrenaline did not lower [Ca(2+)](i) in the presence of 30mM extracellular K(+), indicating that adrenaline does not exert a direct effect on Ca(2+) channels but increases K(+) channel activity. As for primary b-cells, [Ca(2+)](i) oscillations persisted in the presence of closed K(ATP) channels; these also persisted in the presence of thapsigargin, which blocks Ca(2+) uptake into Ca(2+) stores. In contrast, in voltage-clamped cells and in the presence of diazoxide (50mM), which hyperpolarizes the cells by opening K(ATP) channels, [Ca(2+)](i) oscillations were abolished. These results support the hypothesis that [Ca(2+)](i) oscillations depend on functional voltage-dependent Ca(2+) and K(+) channels and are interrupted by a hyperpolarization in insulin-secreting cells.     

Novel effects of propranolol. Release of internal Ca(2+) followed by activation of capacitative Ca(2+) entry in Madin Darby canine kidney cells

The effect of propranolol on Ca(2+) signalling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Propranolol increased cytosolic free Ca(2+) levels ([Ca(2+)](i)) in a concentration-dependent manner between 0.1 and 1 mM. The response was partly inhibited by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with 0.2 mM propranolol partly inhibited the [Ca(2+)](i) rise induced by 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with thapsigargin abolished propranolol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 0.2 mM propranolol in Ca(2+)-free medium. Propranolol (0.2 mM) inhibited 25% of thapsigargin-induced capacitative Ca(2+) entry. Suppression of 1,4,5-trisphosphate (IP(3)) formation by 2 microM U73122, a phospholipase C inhibitor, did not alter 0.2 mM propranolol-induced internal Ca(2+) release. Propranolol (1 mM) also increased [Ca(2+)](i) in human neutrophils. Collectively, we have found that 0.2 mM propranolol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive Ca(2+) stores in an IP(3)-independent manner, followed by Ca(2+) influx from external space. Independently, propranolol was able to inhibit thapsigargin-induced capacitative Ca(2+) entry.     

cGMP stimulates endoplasmic reticulum Ca(2+)-ATPase in vascular endothelial cells

Calcium is a crucial regulator of many physiological processes such as cell growth, division, differentiation, cell death and apoptosis. In this study, we examined the effect of cGMP on agonist-induced [Ca(2+)](i) transient in isolated rat aortic endothelial cells. 100 microM ATP was applied to the cells bathed in a Ca(2+)-free physiological solution to induce a [Ca(2+)](i) transient that was caused by Ca(2+) release from intracellular stores. cGMP, which was applied after [Ca(2+)](i) reached its peak level, accelerated the falling phase of [Ca(2+)](i) transient. Pre-treatment of the cells with CPA abolished the accelerating effect of cGMP on the falling phase of [Ca(2+)](i) transient. The effect of cGMP was reversed by KT5823, a highly specific inhibitor of protein kinase G. Taken together, these data suggest that cGMP may reduce [Ca(2+)](i) level by promoting Ca(2+) uptake through sarcoplasmic/endoplasmic reticulum ATPase and that the effect of cGMP may be mediated by protein kinase G.